Interferon polymer conjugates

ABSTRACT

Compositions containing alpha interferon conjugated to a substantially non-antigenic polymer are disclosed in which at least about 30% of the conjugates include covalent attachment of the alpha interferon to the substantially non-antigenic polymer at a histidine. Also disclosed is a process for preparing the conjugates. The process includes contacting an alpha interferon with a succinimidyl carbonate-activated substantially non-antigenic polymer at a pH which is sufficient to facilitate covalent attachment of the polymer on a histidine of the alpha interferon.

This application is a continuation os U.S. patent application Ser. No.08/994,622, filed on Dec. 19, 1997, which has now issued as U.S. Pat.No. 5,951,974, which, in turn, is a continuation-in-part of U.S. patentapplication Ser. No. 08/337,567, filed Nov. 10, 1994, now U.S. Pat. No.5,711,944 which, in turn, is a continuation-in-part of U.S. patentapplication Ser. No. 08/150,643, filed Nov. 10, 1993, now abandoned. Thecontents of each application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to interferon-polymer conjugates. Inparticular, the invention is directed to conjugates having a novelinterferon-polymer attachment profile.

2. Description of Related Art

Conjugating biologically-active proteins to polymers has been suggestedto improve one or more of the properties of circulating life, watersolubility or antigenicity in vivo. For example, some of the initialconcepts of coupling peptides or polypeptides to polyethylene glycol(PEG) and similar water-soluble polymers are disclosed in U.S. Pat. No.4,179,337, the disclosure of which is incorporated herein by reference.

Insulin and hemoglobin were among the first therapeutic agentsconjugated. These relatively large polypeptides contain several freelysine ε-amino attachment sites. Several polymers could be attachedwithout significant loss of biologic activity.

For many biologically active materials, the conjugation process,however, is not without complications. Care must be taken to limit theloss of biological activity caused by the conjugation reaction. Forexample, if too much of the activated polymer is attached to the targetprotein or polypeptide, biological activity can be severely reduced orlost. Further, if the wrong linker joining the polymer to the protein isused or an insufficient amount of polymer is attached to the target, thetherapeutic value of the resultant conjugate is rather limited. Often,such conjugates do not demonstrate enough of an increase in thecirculating life to compensate for the loss in bioactivity. Problems canalso result when a therapeutic moiety's active site (i.e. where groupsassociated with bioactivity are found) becomes blocked as a result ofthe polymer attachment. This problem can be difficult to avoid since thepolymer and protein are typically joined in solution-based reactions.Pre-blocking the active sites with materials such as pyridoxal phosphatehas been suggested, but the results have been inconsistent. The problemsare particularly acute with lower molecular weight proteins andpeptides. These bioactive materials often have few attachment sites notassociated with bioactivity.

Interferons, hereinafter also referred to as IFN's, are a particularexample of proteins which could benefit from improved polymerconjugation techniques. See, for example, U.S. Pat. Nos. 4,766,106 and4,917,888 which describe inter alia beta interferon conjugated withactivated polymers including mPEG-2,4,6-trichloro-S-triazine,mPEG-N-succinimidyl glutarate or mPEG-N-succinimidyl succinate. Thepatentees disclose that covalent modification of the protein is done ata pH of from 5 to 9 and, when the protein is reacted through its lysineresidues, covalent modification of the protein is done at a pH of from 8to 9. Relatively high molar excesses (10, 20 and 50-fold) of theactivated polymer are also used.

European Patent Application bearing publication No. 0 236 987 describesreacting alpha and gamma interferons with high molar excesses of alkylimido ester-activated polyethylene glycols under conditions whichpreferably include a pH of from approximately 7 to 9. European PatentApplication bearing publication No. 0 510 356 describes conjugatingalpha interferon with pyridinyl carbonyl and thiocarbonyl activated PEGat a pH of from 7 to 9. There was no mention in these disclosures thatamino acids other than lysine were involved in the conjugation or thatit would be advantageous to do so.

In spite of the above-described disclosures, most interferon-polymerconjugates have been deemed to be unacceptable for one reason oranother.

The present invention addresses these shortcomings.

SUMMARY OF THE INVENTION

In one aspect, the present invention includes pharmaceuticalcompositions containing a mixture of mono-polymer stranded alphainterferon conjugates. In the mixture, individual mono-polymer-IFNconjugates are defined as positional isomers, depending upon which aminoacid residue is covalently attached to the polymer. Within this mixtureis an isomer which is an alpha interferon covalently conjugated to apolymer at a histidine residue on the alpha interferon. The compositionsare distinguishable from prior art products in part due to the fact thatat least about 15%, and preferably at least about 30%, of the interferonconjugates included as part of the composition have a polymer covalentlyattached to a histidine of the alpha interferon. Preferably, however,the conjugates or positional isomers contain about one polymer strandper alpha interferon, regardless of where the polymer is attached.

Still further aspects of the invention include methods of preparingalpha-interferon conjugates and compositions prepared by the methods.The IFN-polymer conjugates are prepared by reacting a solutioncontaining alpha interferon with a sufficient amount of anoxycarbonyl-N-dicarboximide-activated polymer such as a succinimidylcarbonate activated PEG under conditions which are sufficient to effectcovalent attachment of the polymer to the interferon, at least in part,to a His residue such as the His34 of alpha interferon. Part of theseconditions include conducting the conjugation reaction within a pH rangewhich is sufficient to facilitate covalent attachment of at least aportion of the polymer strands to histidine residue amino groups of theinterferon molecules.

Suitable alpha-interferons include recombinant and non-recombinantalpha-interferons isolated from mammals. The polymer portion of theconjugate is preferably a polyalkylene oxide (PAO), such as amonomethoxy polyethylene glycol (mPEG). In alternative embodiments,other substantially non-antigenic polymers can also be used. Thepolymers preferably have a molecular weight of from about 200 to about35,000.

The conditions for effecting conjugation include conducting theattachment reaction with from about an equi-molar to about a relativelysmall molar excess of the activated polymer with respect to thealpha-interferon. The conditions further include conducting the reactionat a pH of less than about 7 and preferably at a pH of from about 4.5 toabout 6.8.

The invention also includes methods of treating alpha-interferonsusceptible conditions in mammals. In this aspect, the treatmentincludes administering an effective amount of the composition containingthe IFN conjugates described herein to mammals requiring such therapy.

Thus, the present invention further provides for analpha-interferon-conjugate, including an alpha-interferon havingattached thereto an amount of a substantially non-antigenic polymer sothat the conjugate has a T_(max) at least about 4 times greater thanunmodified alpha-interferon measured under the same conditions, or evena T_(max) that is at least about 8 times greater than unmodifiedalpha-interferon measured under the same conditions.

In an additional feature, the present invention provides for analpha-interferon-conjugate, including an alpha-interferon havingattached thereto an amount of a substantially non-antigenic polymer suchas so that the conjugate has an AUC at least about 10 times that ofunmodified alpha-interferon measured under the same conditions.

In a further additional feature, the conjugate has at least about 10% ofthe activity of unmodified alpha-interferon measured under the sameconditions when the conjugate is administered subcutaneously in amammal, including when the conjugate has an AUC at least about 10 timesthat of unmodified alpha-interferon measured under the same conditions.Optionally, the conjugate also has a T_(1/2), of about 5 hours alphaphase when said conjugate is administered subcutaneously in a mammal,including when the conjugate has an AUC at least about 10 times that ofunmodified alpha-interferon measured under the same conditions.

Preferably, the substantially non-antigenic polymer employed to achieveconjugates with the above-described pharmacokinetic parameters is apolyalkylene oxide, and more preferably, the polyalkylene oxide is apolyethylene glycol.

More preferably, the invention provides for a composition that includesa mixture of alpha-interferon conjugate positional isomers, wherein oneof the positional isomers includes an alpha interferon covalentlyconjugated to a substantially non-antigenic polymer, such as, forexample, an alkyl terminated polyalkylene oxide, at a histidine residueon the alpha interferon, so as to provide the above-describedpharmacokinetic properties.

As a result of the present invention, it has been unexpectedly foundthat additional improvements in interferon-polymer conjugatecompositions are possible. For example, by modifying the conjugationconditions, it is now possible to obtain compositions containingrelatively high activity mono-polymer IFN conjugates in which a portionof the alpha interferon is attached at unique locations to polymers. Inaddition, it has been found that conducting the conjugation reactionwith succinimidyl carbonate and some relatedoxycarbonyl-N-dicarboximide-type activated polymers, such as SC-PEG, atpH levels which are more acidic than that typically used forconjugation, will cause the polymer to attach not only at the expectedlysine sites on the IFN molecule, but also selectively on histidinesites such as the preferred His34 amino acid on alpha interferons.

For a better understanding of the present invention, reference is madeto the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a series of chromatograms referred to in Example 11.

FIG. 2 is a series of chromatograms referred to in Example 13.

DETAILED DESCRIPTION OF THE INVENTION

1. Interferons

The interferon (IFN) portion of the polymer conjugate can be prepared orobtained from a variety of sources including recombinant techniques suchas those using synthetic genes expressed in E. coli. See also Pestka,"Interferon α" in Human Cytokines, Black-well Scientific Publications1-16 (1992), the disclosure of which is incorporated herein byreference. In addition, the IFN can also be a mammalian source extractsuch as human, ruminant or bovine αIFN. One particularly preferred IFNis IFNα-2b, a recombinantly-made product of the Schering Corp.,Kenilworth, N.J.

The term "interferon" or "IFN" as used herein means the family of highlyhomologous species-specific proteins that inhibit viral replication andcellular proliferation and modulate immune response. Human interferonsare grouped into three classes based on their cellular origin andantigenicity: α-interferon (leukocytes), β-interferon (fibroblasts) andγ-interferon (B cells). Recombinant forms of each group have beendeveloped and are commercially available. Subtypes in each group arebased on antigenic/structural characteristics. At least 24 interferonalphas (grouped into subtypes A through H) having distinct amino acidsequences have been identified by isolating and sequencing DNA encodingthese peptides. See also Viscomi, 1996 Biotherapy 10:59-86, the contentsof which are incorporated herein by reference. The terms "α-interferon","alpha interferon", "interferon alpha" and "human leukocyte interferon"are used interchangeably in this application to describe members of thisgroup. Both naturally occurring and recombinant α-interferons, includingconsensus interferon such as that described in U.S. Pat. No. 4,897,471,the contents of which are incorporated herein by reference, may be usedin the practice of the invention.

The purification of interferon alpha from human leukocytes isolated fromthe buffy coat fraction of whole blood is described in U.S. Pat. No.4,503,035. Human leukocyte interferon prepared in this manner contains amixture of human leukocyte interferons having different amino acidsequences. Purified natural human α-interferons and mixtures thereofwhich may be used in the practice of the invention include but are notlimited to Sumiferon® interferon alpha-nI available from Sumitomo,Japan, Wellferon® interferon alpha-nI (Ins) available fromGlaxo-Wellcome Ltd., London, Great Britain, and Alferon® interferonalpha-no available from the Purdue Frederick Co., Norwalk, Conn.

The advent of recombinant DNA technology applied to interferonproduction has permitted several human interferons to be successfullysynthesized, thereby enabling the large-scale fermentation, production,isolation, and purification of various interferons to homogeneity.Recombinantly produced interferon retains its in vitro and in vivoantiviral and immunomodulatory activities. It is also understood thatthe recombinant techniques could also include a glycosylation site foraddition of a carbohydrate moiety on the recombinantly-derivedpolypeptide.

The construction of recombinant DNA plasmids containing sequencesencoding at least part of human leukocyte interferon and the expressionin E. coli of a polypeptide having immunological or biological activityof human leukocyte interferon is disclosed in U.S. Pat. No. 4,530,901and European Patent No. EP 0 032 134. The construction of hybrida-interferon genes containing combinations of different subtypesequences (e.g., A and D, A and B, A and F) is disclosed in U.S. Pat.Nos. 4,414,150, 4,456,748 and 4,678,751. Typical suitable recombinantα-interferons which may be used in the practice of the invention includebut are not limited to interferon alpha-2b such as Intron® A availablefrom Schering Corporation, Kenilworth, N.J., interferon alpha-2a such asRoferon® A available from Hoffmann-La Roche, Nutley, N.J., and Infergen®available form Amgen, Thousand Oaks, Calif.

Alternate embodiments, where the foreign αIFN is not completelyautologous, may be also used if desired. A key, however, is that thenon-autologous αIFN has sufficient bioactivity or αIFN effect such asantiviral activity in the target mammal. Other substances including αIFNfractions or predecessor polypeptides can also be included in theconjugates of the present invention. As used herein, "α-IFN effect inmammals" means in vivo activity corresponding to that observed withαIFN's. These substances are prepared by using techniques known to thoseof ordinary skill in the art such as tissue culture, extraction fromanimal sources or by recombinant DNA methodologies. Transgenic sourcesof αIFN and related moieties are also contemplated. Such materials areobtained from transgenic animals, i.e. mice, pigs, cows, etc. where theαIFN protein is expressed in milk, blood, or other tissues. The methodby which the αIFN is prepared for the conjugates of the presentinvention is not limited to those described herein. For purposes of thepresent invention, the αIFN's are preferred because of their biochemicaland serological properties. In particular, αIFN has documented antiviralproperties and diffuses more effectively into the bloodstream than otherinterferons.

2. Non-Antigenic Polymers

To conjugate the IFN to polymers such as poly(alkylene oxides), one ofthe polymer hydroxyl end-groups is converted into a reactive functionalgroup which allows conjugation. This process is frequently referred toas "activation" and the product is called an "activated" polymer oractivated poly(alkylene oxide). Other substantially non-antigenicpolymers are similarly "activated" or functionalized.

The activated polymers are reacted with αIFN so that attachment occursat ε-amino groups of lysines, the N-terminal cysteine amino group and,as described below, at amino groups on histidines. Free carboxylic acidgroups, suitably activated carbonyl groups, oxidized carbohydratemoieties and mercapto groups if available on the IFN can also be used assupplemental attachment sites, if desired.

In a preferred aspect of the invention, urethane (carbamate) linkagesare formed between one of the αIFN amino acid amino groups (i.e. lysine,histidine, N-terminal), and the activated polymer. Preferably, theurethane linkage is formed using a terminaloxycarbonyl-oxy-N-dicarboximide group such as a succinimidyl carbonategroup. Alternative activating groups include N-succinimide,N-phthalimide, N-glutarimide, N-tetrahydrophthalimide andN-norborene-2,3-dicarboxide. These urethane-forming groups are describedin commonly owned U.S. Pat. No. 5,122,614, the disclosure of which ishereby incorporated by reference. This patent also discloses theformation of N-succinimide carbonate derivatives of polyalkylene oxidesincluding polyethylene glycols which are also capable of formingurethane linkages with lysine amino group targets.

Among the substantially non-antigenic polymers, mono-activated,alkoxy-terminated polyalkylene oxides (PAO's), such asmonomethoxy-terminated polyethylene glycols (mPEG's) are preferred;bis-activated polyethylene oxides (glycols) are also contemplated forpurposes of cross-linking αIFN's or providing a means for attachingother moieties such as targeting agents for localizing the polymer-αIFNconjugate in a particular area such as, for example, the liver.

Suitable polymers will vary substantially by weight. Polymers havingmolecular number average weights ranging from about 200 to about 35,000are usually selected for the purposes of the present invention.Molecular weights of from about 1,000 to about 15,000 are preferred and2,000 to about 12,500 are particularly preferred.

The polymeric substances included are also preferably water-soluble atroom temperature. A non-limiting list of such polymers includepolyalkylene oxide homopolymers such as polyethylene glycol (PEG) orpolypropylene glycols, polyoxyethylenated polyols, copolymers thereofand block copolymers thereof provided that the water solubility of theblock copolymers is maintained. In addition to mPEG, C₁₋₄alkyl-terminated polymers are also useful.

As an alternative to PAO-based polymers, effectively non-antigenicmaterials such as dextran, polyvinyl pyrrolidones, polyacrylamides suchas HPMA's-hydroxypropylmethacrylamides, polyvinyl alcohols,carbohydrate-based polymers, copolymers of the foregoing, and the likecan be used. Those of ordinary skill in the art will realize that theforegoing list is merely illustrative and that all polymer materialshaving the qualities described herein are contemplated. For purposes ofthe present invention, "substantially or effectively non-antigenic"means all materials understood in the art as being nontoxic and noteliciting an appreciable immunogenic response in mammals.

3. Reaction Conditions

Conjugation reactions, sometimes referred to as PEGylation reactions,are often carried out in solution without regard to where the polymerwill attach to the protein. Such techniques are also usually carried outat slightly alkaline i.e. pH 7+ to about 9 for conjugating αIFNs. A keyto the present invention, however, is that the retained IFN bioactivitycan be maximized if the polymer is attached to a histidine, preferablyHis34 on IFNα 2b. It will be appreciated by the artisan that althoughvarious species of the αIFN may or may not have a histidine at aminoacid 34, the interferon conjugates will nonetheless preferably includeat least some positional isomers containing a polymer attached at anavailable histidine.

The processes of the present invention therefore includes reacting asolution containing an alpha interferon with an amount of anoxycarbonyl-oxy-N-dicarboximide-activated polymer such as succinimidylcarbonate-activated MPEG at a pH which is sufficient to facilitatecovalent attachment of at least a portion of the polymer strands to ahistidine, such as the His34 of IFNα2b, of the individual interferonmolecules. In particular, the pH will preferably be slightly acidic,i.e. less than about 7.0; more preferably, less than about 6.8 and mostpreferably in the range of from about 4.5 to about 6.8.

The reaction conditions for effecting conjugation further includeconducting the attachment reaction with from about equi-molar to about arelatively small molar excess of the activated polymer with respect tothe alpha-interferon. In this regard, the process can be carried outwith about 1-8-fold molar excesses; preferably about 1.5-7-fold molarexcesses and most preferably about 1.75-5-fold molar excesses. Theconjugation reaction can be carried out at about room temperature,20-25° C. It is also preferred that the coupling reaction be allowed toproceed for rather short periods of time, i.e. 1-2 hours, beforequenching. In practice, the reaction conditions provide a mixture ofpolymer-IFN positional isomers. Preferably, each isomer contains asingle polymer strand attached to the interferon via an amino acidresidue. In alternative embodiments, there can be more than one strandof polymer attached as a result of the process. Solutions containingthese conjugates are also useful as is or can be further processed toseparate the conjugates on the basis of molecular weight.

Characterization of the preferred one polymer strand-IFN conjugates(isomers) via cation exchange chromatography into separated peaksrevealed that the polymer can be attached at up to about eight differentsites on the IFNα2b molecule. These sites, representing individualpositional isomers, are Cys1, Lys31, His34, Lys49, Lys83, Lysl21,Lys131, Lys134. In some preferred embodiments, the reaction poolscontaining mono-polymer-IFN conjugates can contain relatively highproportions of the His34 positional isomer, i.e. about 30-60%, the Cys1positional isomer, about 7-20%, and the Lys121 positional isomer, about7-15%, with the rest of the positional isomers comprising the remainder.It will be understood that alternative IFN's will provide alternativedistributions of positional isomers, depending upon the amino acidsequence of the starting material.

Due to the nature of the solution-based conjugation reactions, thecompositions are a heterogeneous mixture of species which contain thepolymer strand(s) attached at different sites on the interferonmolecule. In any solution containing the conjugates, it is likely that amixture of at least about 3, preferably about 6 and more preferablyabout 8 positional isomers will be present. For example, when IFNα2b isused, the solution will contain conjugate isomers with the polymerattached at one or more of Cys1, Lys31, His34, Lys49, Lys83, Lys121,Lys131, and Lys134 of the interferon. In the case of IFNα2b and thepreferred forms of activated polymers described herein, the 3 mostprominent sites of attachment are His34 (55%), Cys1 (15%) and Lys121(15%).

A preferred composition of the invention is a mixture of the IFN-polymerisomers which are composed of at least about 15% His-polymersubstituted-IFN. That is, at least about 15% of the conjugates includecovalent attachment of the alpha interferon to the substantiallynon-antigenic polymer at a His. In more preferred aspects, at leastabout 30%, and in most preferred aspects of the invention, at leastabout 40% of the conjugates include the His34 covalent polymerattachment. When IFNα2b or related IFN's are used, the histidineattachment site is preferably His34.

4 Effect of Reaction pH Upon PEG-IFN Positional Isomers Distribution

The process of the present invention takes advantage of the discoverythat the site of polymer attachment on alpha interferon is influenced toa large extent by the pH of the reaction system. As the pH of thereaction solution is varied, the reactivity towards specific forms ofactivated polymers of the various functional groups such asalpha-amines, imidazoles and epsilon amines will vary. Typically,polymer conjugation reactions are carried out at basic pHs in order tomaximize attachment at lysine epsilon amino groups. For example,Zalipsky et al. Biotech & App. Biochem Vol 15, p. 100-114; (1992)evaluated the SC-PEG reagent for PEGylation and reported that theoptimal reactivity was at about pH 9.3. The method of the presentinvention, however, includes conducting the reaction at lower pH's inorder to allow a portion of the activated polymer strands to attach tohistidine amino groups and de-emphasize, but not eliminate, lysine sitesfor attachment.

Furthermore, it has also been found that the biological activity of thevarious polymer conjugate positional isomers unexpectedly differs, evenwhen each of the positional isomers has the same degree of polymersubstitution.

The method described herein affords novel attachment of polymers such asPEG to a specific histidine residue in IFN molecules. In preferredembodiments, the conjugation reaction results in a substantial amount,i.e. at least about 30% of the conjugates being linked at IFN histidinesites such as the His34 on IFNα2b.

It has also been unexpectedly determined that the relative distributionof the positional isomers is largely dependent upon the pH at which theconjugation reaction is carried out. Shifting the pH from basic toslightly acidic pH (5.5-6.5) favors the formation of conjugates linkedat His34 on IFNα2b, and to a lesser extent, the N-terminus (Cys1). UsingpH(8-10) during the conjugation reaction, on the other hand, favors theformation of lysine-related attachment sites, confirmed via cationexchange chromatography. In those situations where IFNα2b is notincluded, the His34 site, of course, may not always be present. Thereaction conditions nonetheless allow covalent attachment of anactivated polymer to a His. Thus, Applicants have demonstrated that thepH of the reaction system influences the placement of some types ofactivated polymers on a protein surface, especially with regard todifferent amino acid residues (i.e. lysine vs. N-terminal amine vs.histidine).

5. Fractionation of Conjugates

Although the inventive process produces a substantial amount ofconjugates having a single polymer strand, conjugates having varyingdegrees of polyalkylene oxide substitution are also generated. Residualunconjugated PAO's and αIFN can also be present. This mixture istypically in a reaction buffer containing one or more of phosphate,chloride and bicarbonate anions. The PAO, αIFN and conjugate mixture ispreferably fractionated in a buffer solution containing from about 1-10mg/ml PAO-αIFN conjugates. Suitable fractionating solutions have a pH offrom about 7.0 to about 9.0 and preferably from about 7.5 to about 8.5.The solutions preferably contain one or more buffer salts selected fromKCl, NaCl, K₂ HPO₄, KH₂ PO₄, Na₂ HPO₄, NaH₂ PO₄, NaHCO₃, NABO₄, (NH₄)₂CO₃ and glycine NaOH. Sodium phosphate buffers are preferred.

Depending upon the reaction buffer, the αIFN-polymer conjugatecontaining solution may first have to undergo bufferexchange/ultrafiltration. For example, the αIFN conjugate solutions canbe ultra filtered across a low molecular weight cut-off (10,000 to30,000 Dalton) membrane which will also remove most surfactants, ifpresent, as well.

The fractionation of the conjugates into desired species is preferablycarried out using an anion exchange medium. Such media are capable ofselectively binding those αIFN-polymer conjugates having 1-4 polymerstrands, excess polymer and unmodified αIFN. This fractionation occurssince the αIFN molecules of various degrees of substitution will haveisoelectric points which vary in a somewhat predictable fashion. Forexample, the isoelectric point of αIFN is determined by the number ofavailable amino groups available on the surface of the protein. Theseamino groups also serve as the point of attachment of polyalkylene oxideconjugates. Therefore, as the degree of substitution of polyalkyleneoxide increases, the isoelectric point decreases, and the ability of theconjugate to bind to an anion exchange resin weakens.

The use of strongly polar anion exchange resins is especially preferredfor the method of the present invention. For this reason, quaternaryamine coated anion exchange resins are utilized. The quaternary amineresin may be coated onto either a polymeric or silica matrix; however,polymeric matrices are preferred. A number of tetramethylamine, orquaternary methylamine, anion exchange resins are commerciallyavailable, coated onto the support matrices. Included among thecommercially available quaternary anion exchange resins suitable for usewith the present invention are Q-HD available from Bio-Sepra; QATRISACRYL® and QMA-SPHEROSIL®, quaternary amine resins coated onto apolymer matrix, manufactured by IBF of Garenne, France, for Sepracor,Inc. of Marlborough, Mass.; TMAE650M®, a tetramethylamino ethyl resincoated onto a polymer matrix, manufactured by EM-Separators ofGibbstown, N.J.; QAE550C®, and SUPERQC®, each a quaternary amine resincoated onto a polymer matrix and manufactured by TosoHaas ofMontgomeryville, Pa. QMA Accell, manufactured by Millipore of Millford,Mass. and PEI resins manufactured by JT Baker of Phillipsburg, N.J., mayalso be used.

The anion exchange resin is packed in the column and equilibrated byconventional means. A buffer having the same pH and osmolality as theconjugated αIFN solution is used. The conjugate-containing solution isthen adsorbed onto the column. At the completion of the loading, agradient flow of an elution buffer with increasing salt concentrationsis applied to the column to elute the desired fractions of polyalkyleneoxide-conjugated αIFN. The fractions are of essentially uniformmolecular weight and degree of substitution.

Preferred IFN conjugate fractions have 14 polymer strands per αIFNmolecule. More preferably, the fraction contains about 1-2 and, mostpreferably, about 1 polymer strand per αIFN molecule. The elution bufferpreferably contains one or more salts selected from KCl, NaCl, K₂ HPO₄,KH₂ PO₄, NAHPO₄, NaH₂ PO₄, NaHCO₃, NaBO₄ and (NH₄)₂ CO₃. These fractionsare substantially free of other conjugates. Any unconjugated species canthen be backwashed from the column by conventional techniques.

Techniques utilizing multiple isocratic steps of increasingconcentration can also be used. Multiple isocratic elution steps ofincreasing concentration will result in the sequential elution ofαIFN-polymer conjugates. The degree of polymer conjugation within eachfraction will be substantially uniform. However, the degree of polymerconjugation for each fraction will decrease with elution time. Ionexchange purification of the conjugates can also be carried out with,for example, a Q-HD Column from Sepracor, Inc. along with a dilutesodium phosphate solution (10 mM NAPO₄ ion). The sample is washed with10 mM NaPO₄ to remove any unreacted PAO and thereafter a step gradientelution with NaCl is used. Elution with 10 mM NaCl recovers fractionscontaining conjugates with greater than 3 polymer strands PAO per IFN;elution with 50 mM NaCl recovers conjugates containing 1-2 strands;elution with 150 mM NaCl recovers unmodified EFN.

The temperature range for elution is between about 4° C. and about 25°C. Preferably, elution is carried out at a temperature of from about 6°C. to about 22° C. The elution of the PAO-αIFN fraction is detected byUV absorbance at 254 nm. Fraction collection may be achieved throughsimple time elution profiles. The preferred fractions can also be pooledin the elution buffer.

6. Surfactants

In another preferred aspect, the reaction conditions include thepresence of a surfactant. The surfactants used in the processes of thepresent invention are ionic-type agents. One particularly preferredagent is sodium dodecyl sulfate, (SDS). Other ionic surfactants such aslithium dodecyl sulfate, quaternary ammonium compounds, taurocholicacid, caprylic acid, decane sulfonic acid, etc. can also be used.Non-ionic surfactants can also be used. For example, materials such aspolyoxyethylene sorbitans (TWEEN®s), polyoxyethylene ethers (Tritons)can be used. See also Neugebauer, A Guide to the Properties and Uses ofDetergents in Biology and Biochemistry (1992) Calbiochem Corp. The onlylimitations on the type of surfactant used in the processes of theinvention are that they do not cause substantial denaturation of the IFNand do not completely inhibit polymer conjugation. The surfactants arepresent in the reaction mixtures in amounts from about 0.01-0.5%;preferably from 0.05-0.5%; and most preferably from about 0.075-0.25%.Mixtures of the surfactants are also contemplated.

7. Pharmacokinetic Parameters

As pointed out above, compositions of the present invention contain aheterogeneous mixture of polymer-IFN species in which the polymerstrand(s) is/are attached at different sites on the interferon molecule.In spite of the heterogeneous nature of the conjugates, the compositionshave a predictable in vivo pharmacokinetic profile which maximizes thetherapeutic effect of the interferon.

Compositions of the present invention containing IFNα preferably includeat least about 15% polymer-His conjugates, more preferably at leastabout 30% and most preferably at least about 40% polymer-His conjugates.While Applicants are not bound by theory, it is believed that thelinkage for the His-positional isomers included in the compositions ofthe invention is relatively labile vis a vis that of the Lys-positionalisomers. As a result, at physiologic pH, the compositions demonstrate arelatively smooth onset on activity after administration as well as aprolonged duration of effect. This profile allows the artisan toadminister the composition in less frequent doses than with unmodifiedIFN's.

8. Methods of Treatment

Another aspect of the present invention provides methods of treatmentfor various medical conditions in mammals, preferably humans. Themethods include administering an effective amount of an αIFN-polymerconjugate containing composition which has been prepared as describedherein to a mammal in need of such treatment. The conjugates are usefulfor, among other things, treating interferon-susceptible conditions orconditions which would respond positively or favorably as these termsare known in the medical arts to interferon-based therapy.

Conditions that can be treated in accordance with the present inventionare generally those that are susceptible to treatment with interferonalpha. For example, susceptible conditions include conditions whichwould respond positively or favorably as these terms are known in themedical arts to interferon alpha-based therapy. For purposes of theinvention, conditions that can be treated with interferon alpha therapyinclude those conditions in which treatment with an interferon alphashows some efficacy, but which may not be treatable with interferonalpha because the negative side effects outweigh the benefits of thetreatment. For example, side effects accompanying alpha therapy havevirtually ruled out treatment of Epstein Barr virus using interferonalpha. Practice of the invention results in substantially reduced oreliminated side effects as compared to conventional interferon alphatreatment.

Exemplary conditions which can be treated with interferon include butare not limited to cell proliferation disorders, in particular cancer(e.g., hairy cell leukemia, Kaposi's sarcoma, chronic myelogenousleukemia, multiple myeloma, basal cell carcinoma and malignant melanoma,ovarian cancer, cutaneous T cell lymphoma), and viral infections.Without limitation, treatment with interferon may be used to treatconditions which would benefit from inhibiting the replication ofinterferon-sensitive viruses. Viral infections which may be treated inaccordance with the invention include hepatitis A, hepatitis B,hepatitis C, other non-A/non-B hepatitis, herpes virus, Epstein-Barrvirus (EBV), cytomegalovirus (CMV), herpes simplex, human herpes virustype 6 (HHV-6)), papilloma, poxvirus, picornavirus, adenovirus,rhinovirus, human T lymphotropic virus-type 1 and 2 (HTLV-1/-2), humanrotavirus, rabies, retroviruses including human immunodeficiency virus(HIV), encephalitis and respiratory viral infections. The method of theinvention can also be used to modify various immune responses.

Variants of interferon alpha are currently approved in the United Statesand other countries for the treatment of hairy cell leukemia, venerealwarts, Kaposi's Sarcoma, and chronic non-A/non-B hepatitis: interferonalpha-2b, marketed under the trade name INTRON® A (Schering Corporation,Kenilworth N.J.), and interferon alpha-2a, marketed under the trade nameRoferon® A (Hoffmann-La Roche, Nutley, N.J.), and consensus interferonmarketed under the trade name Infergen™ (Amgen, Thousand Oaks, Calif.).Since interferon alpha-2b, among all interferons, has the broadestapproval throughout the world for treating chronic hepatitis Cinfection, it is most preferred for use in the treatment of chronichepatitis C in accordance with practice of the invention.

Administration of the described dosages may be every other day, but ispreferably once or twice a week. Doses are usually administered over atleast a 24 week period by injection.

Administration of the dose can be intravenous, subcutaneous,intramuscular, or any other acceptable systemic method. Based on thejudgment of the attending clinician, the amount of drug administered andthe treatment regimen used will, of course, be dependent on the age, sexand medical history of the patient being treated, the neutrophil count(e.g. the severity of the neutropenia), the severity of the specificdisease condition and the tolerance of the patient to the treatment asevidenced by local toxicity and by systemic side-effects. Dosage amountand frequency may be determined during initial screenings of neutrophilcount.

Conventional pharmaceutical formulations can be also prepared using theconjugate-containing compositions of the present invention. Theformulations comprise a therapeutically effective amount of theinterferon-polymer conjugate composition together with pharmaceuticallyacceptable carriers. For example, adjuvants, diluents, preservativesand/or solubilizers, if needed, may be used in the practice of theinvention. Pharmaceutical compositions of interferon including those ofthe present invention may include diluents of various buffers (e.g.,Tris-HCl, acetate, phosphate) having a range of pH and ionic strength,carriers (e.g., human serum albumin), solubilizers (e.g.,Polyoxyethylene Sorbitin or TWEEN®, polysorbate), and preservatives(e.g., thimerosol, benzyl alcohol). See, for example, U.S. Pat. No.4,496,537.

The amount of the α-IFN polymer conjugate administered to treat theconditions described above is based on the IFN activity of the polymericconjugate. It is an amount that is sufficient to significantly affect apositive clinical response. Although the clinical dose will cause somelevel of side effects in some patients, the maximal dose for mammalsincluding humans is the highest dose that does not cause unmanageableclinically-important side effects. For purposes of the presentinvention, such clinically important side effects are those which wouldrequire cessation of therapy due to severe flu-like symptoms, centralnervous system depression, severe gastrointestinal disorders, alopecia,severe pruritus or rash. Substantial white and/or red blood cell and/orliver enzyme abnormalities or anemia-like conditions are also doselimiting.

Naturally, the dosages of the various αIFN compositions will varysomewhat depending upon the αIFN moiety and polymer selected. Ingeneral, however, the conjugate is administered in amounts ranging fromabout 100,000 to about several million IU/m² per day, based on themammal's condition. The range set forth above is illustrative and thoseskilled in the art will determine the optimal dosing of the conjugateselected based on clinical experience and the treatment indication.

The pharmaceutical compositions may be in the form of a solution,suspension, tablet, capsule, lyophilized powder or the like, preparedaccording to methods well known in the art. It is also contemplated thatadministration of such compositions will be chiefly by the parenteralroute although oral or inhalation routes may also be used depending uponthe needs of the artisan.

EXAMPLES

The following examples serve to provide further appreciation of theinvention but are not meant in any way to restrict the effective scopeof the invention.

Example 1 Preparation of rαIFN-PEG₅,000 in Presence of SDS (0.1%)

In this example, recombinant αIFN-2b, (rαIFN), a product of theSchering-Plough Corporation, Kenilworth, N.J. was conjugated withactivated polyethylene glycol-N-succinimide carbonate (SC-PEG) asdescribed in U.S. Pat. No. 5,122,614. The polymer had a molecular weightof about 5,000.

36 mg of the rαIFN was dialyzed into 0.1 molar sodium phosphate pH 7.5using a Centricon-10 (a product of the Amicon Corporation of Beverly,Mass.). The final concentration of rαIFN was about 3 mg/ml. 0.1 ml of10% SDS was added to the rαIFN and was allowed to incubate at roomtemperature for 10 minutes. Thereafter, 42 mg of SC-PEG₅,000 was addedto the protein-SDS solution and stirred at room temperature for twohours and then quenched with glycine. Next, the reaction mixture wasdialyzed into 10 mM sodium phosphate pH 8 to fractionate the PEGylatedIFN using a Centricon-30.

Example 2 Preparation of rαIFN-PEG₁₂,000 in presence of SDS (0.1%)

In this Example, the steps of Example 1 were repeated except that thepolyethylene glycol had a molecular weight of about 12,000. Reactionsteps were exactly the same to provide the PEG₁₂,000 conjugate.

Example 3 Fractionation of 2PEG₅,000 rαIFN

In this Example the conjugates prepared in accordance with Example 1were fractionated to obtain the desired 2-PEG₅,000 fraction. ThePEG-αIFN in sodium phosphate buffer was loaded onto a QHD anion exchangecolumn. The 2-PEG fraction was eluted with a gradient from 0 to 400 mMsodium chloride in 10 mM phosphate pH 8. The 2-PEG fraction was verifiedusing size exclusion chromatography and SDS-PAGE.

Example 4 Fractionation of 2PEG₁₂,000 rαIFN

The polymer conjugates of Example 2 were fractionated in the mannerdescribed in Example 3 and verified in the same manner.

Examples 5-8

In these examples, additional preparations of PEG₁₂,000 -rαIFN wereprepared as described previously except that no surfactant was used.Following the conjugation reactions, the samples were tested forretained activity and PEG number. The results are provided below in thetable.

                  TABLE 1                                                         ______________________________________                                        IFN-PEG.sub.12,000                                                                           ACTIVITY (CPE)                                                 PREPARATION    % OF CONTROL PEG #                                             ______________________________________                                        Example 6      26           1.2                                               Example 7      26           1.3                                               Example 8      24           1.0                                               ______________________________________                                    

Example 9 Comparative Data

In this example, the product of Example 3, (SDS-2-PEG-₅,000 rαIFN),2-PEG₅,000 rαIFN made in the absence of a surfactant and unconjugatedrαIFN were tested. Activity was determined using a CPE assay with EMCvirus challenging A549 human lung carcinoma cells. Circulating life wasdetermined using an average value obtained from the blood of 3 rats in agroup receiving 1 million units, with time points taken over 7 days.

                  TABLE 2                                                         ______________________________________                                                          VIRAL      CIRCULATING                                                        PROTECTION HALF LIFE                                                 ACTIVITY ASSAY IC.sub.50                                                                          α PHASE                                             (%)      (pg/ml)    (HRS.)                                           ______________________________________                                        A.  IFN-SDS    69         2.2      5.8                                            2-PEG.sub.5,000                                                           B.  IFN-PEG.sub.5,000                                                                        30         4.0      6.8                                        C.  IFN        100        1.5      0.17                                       ______________________________________                                    

This data clearly shows the advantages of the inventive process.Retained activity is over twice as great as that obtained using standardtechniques.

Example 10

In this example, various pharmacokinetic data was generated using2PEG-rαIFN conjugates prepared according to the methods described above.These samples were compared to unmodified IFN according to the protocolset out in Table 4. Sample B was prepared with SDS.

                  TABLE 3                                                         ______________________________________                                        Retained Activity                                                                         PEG MOLECULAR                                                                              CPE ACTIVITY                                         SAMPLE      WEIGHT       (% CONTROL)                                          ______________________________________                                        A           5,000        35                                                   B           5,000        69                                                   C           12,000       26                                                   D           12,000       26                                                   ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Pharmacokinetic Protocol                                                      ______________________________________                                        ANIMALS:  Sprague Dawley (3 rates/time point)                                 DOSE:     10 × 10.sup.6 UN IFN/rat                                      ROUTE:    Subcutaneous (S.C.)                                                 DRUG:     2-PEG-IFNα's 5,000 and 12,000 mol. wt. PEG                    TIME POINTS:                                                                            0 min., 5 min., 15 min., 30 min., 1 hr., 2 hr., 4 hr.,                        8 hr., 24 hr., 48 hr., 5 days, and 7 days following drug                      administration.                                                     ASSAY:    CPE Assay using serum samples in an EMC virus and                             A549 human lung carcinoma.                                          ______________________________________                                    

Tables 5 and 6 Summary of Pharmacokinetics Data for PEG-Interferons

                  TABLE 5                                                         ______________________________________                                               IC.sub.50       % AC-           Cmax                                   SAMPLE (pg/ml)         TIVITY   AUC    (IU/ml)                                ______________________________________                                        NATIVE 1.52     pg/ml (N = 6)                                                                            100%   145,720                                                                              60,000                               IFNα                                                                    A      4.0      pg/ml (N = 3)                                                                            35%    348,920                                                                              24,433                               B      2.2 ± 0.5                                                                           pg/ml (N = 3)                                                                            69%    351,037                                                                              --                                   C      5.8 ± 2.2                                                                           pg/ml (N = 3)                                                                            26%    1,574,682                                                                            62,750                               ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        SAMPLE   T.sub.max (hr)                                                                          T.sub.1/2 α PHASE (hr)                                                               T.sub.1/2 β PHASE (HR)                   ______________________________________                                        NATIVE IFNα                                                                      1         0.17         --                                            A        4         6.8          48                                            B        2-3       5.8          --                                            C        8         12.1         33                                            ______________________________________                                    

The foregoing data provide the following conclusions:

2-PEG-rαIFN conjugates prepared with both 5,000 and 12,000 molecularweight have distinct advantages over unmodified interferon in mammals.In the case of subcutaneously administered compositions, T_(max) issubstantially increased by the conjugation of the protein with about 2PEG's. For chronic conditions, longer T_(max) 's are desirable and allowclinicians to space out recurring administrations due to the lengtheningof the duration of effect. Even more unexpected, however, was the factthat 2-PEG₁₂,000 conjugates are able to unexpectedly increase AUC byover 10-fold. This dramatic increase in area under the curve was notproportional to the additional polymer weight. Clearly, therapeuticadvantages are realized by this unexpected increase.

Example 11 Effect of pH on PEGylation

In order to probe this effect, the polymer conjugation (PEGylation)reaction of Examples 5-8 was repeated using mPEG₁₂,000 (no surfactant)at four different pHs, 5.4, 6.5, 8.0 and 10.0. The ratio of 2.6 grams ofSC-PEG₁₂,000 to 1 gram of IFN (molar ratio 3.9:1) was used for thereactions at pH 5.4, 6.5 and 8.0 while the ratio of 2.1 grams of 25SC-PEG₁₂,000 to 1 gram of IFN (molar ratio 3.2:1) was used at pH 10. Atthe end of the reaction, glycine was added to quench any residualPEGylation reagent. The product from each reaction was then purifiedusing a Q-hyper D resin at pH 8 with salt elution to remove unreactedingredients.

The purified conjugates obtained at the different pHs were evaluated fortheir biological activity, hydroxylamine sensitivity and distribution ofpositional isomers. Biological activity was determined by specificactivity (MTT-CPE assay).

Hydroxylamine sensitivity was undertaken to determine what percentage ofthe conjugates were PEGylated at histidine sites, including theIFN-His34. Hydroxylamine is a known reagent that we have found toselectively cleave PEG from IFN histidines. An aliquot of each of thesamples (50 μl) was diluted with 0.45 ml of 10 mM sodium phosphate pH7.0. An aliquot of this protein solution (150 μl) was treated with 150μl of 0.5 M hydroxylamine and incubated at room temperature for 60minutes. Thereafter, a volume of 75 ill was loaded on a Mini-S column(Pharmacia Biotech) for cation exchange chromatography. Mobile phase Aincluded 10 mM sodium acetate pH 5.3 buffer and 25% 2-propanol. Mobilephase B contained 500 mM sodium chloride dissolved in mobile phase A.The flow rate was set at 0.5 ml/min and the eluted protein was detectedat 214nm. The individual PEG-IFN solutions were diluted with 10 mMsodium acetate pH 5.3, containing 2-propanol (5%) to 1 mg/ml proteinconcentration. Injection volumes ranged from 10 to 30 μl, depending uponthe protein concentration. A linear gradient was used. The results areset forth in the Table 7 below and in FIG. 1.

FIG. 1 shows the overlay of the chromatograms obtained from the Mono-Scation exchange chromatography column of the different pH reactionproducts. The site of polymer conjugation for each positional isomer wasdetermined by digestion of individual peaks from cation exchangechromatography using proteolytic enzymes (trypsin, V8-protease,chymotrypsin or subtilisin), isolation of PEGylated fragments, andanalysis by N-terminal sequencing and mass spectroscopy.

As seen in the figure, the distribution of the positional isomerschanges significantly as the pH of the reaction changes. The higher thepH, the less His34-linked PEG-IFN and, less dramatically, Cys1-linkedPEG-IFN products are produced.

Table 7 summarizes the specific bioactivity as determined using theMTT-CPE bioassay for IFN and the amount of IFN released upon treatmentwith 0.5M hydroxylamine for 2 hours at 25° C. for the differentconjugate products. These findings confirm that the differences seen inFIG. 1 can also be related to different biological characteristics ofthe products. When the conjugation is conducted at a higher pH (i.e. 8or 10) the products formed are less bioactive and more resistant tohydroxylamine, which therefore means that at higher pH's, less polymeris on His34.

                  TABLE 7                                                         ______________________________________                                        Bioactivities and Hydroxylamine Sensitivities of PEG-IFNs Generated at        Different pHs                                                                             Specific Activity                                                                         % of Conjugate                                                    (CPE assay) Converted to IFN by                                   Reaction pH MIU/mg      Hydroxylamine                                         ______________________________________                                        5.4         61.8        56%                                                   6.5         74.5        47%                                                   8.0         33.3         8%                                                   10.0        27.8        <1%                                                   ______________________________________                                    

The above results indicate that pH is a key variable of the conjugationreaction and that the relative distribution of the positional isomersvaries dramatically with pH. Unexpectedly, the bioactivity of theresultant PEG-IFN mixture of positional isomers is also affected.

Example 12 Comparison of Urethane Linkage Forming Activated Polymers

In this example, effect of pH on reaction conditions was compared usinga different type of urethane linker to see if the activating group hadany role in determining the site of polymer attachment and bioactivity.In particular, the Methoxypoly(ethylene glycol)-succinimidyl carbonateMW 12,000 (SC-PEG₁₂,000) used in the earlier examples was compared withmethoxypoly(ethylene glycol)-2-pyridyl carbonate, MW 12,000(PC-PEG₁₂,000) disclosed in U.S. Pat. No. 5,382,657, as the activatedpolymer reagents for interferon alpha-2b (IFN). The conjugationreactions were carried out for both reagents, SC-PEG₁₂,000 andPC-PEG₁₂,000, at pH 6.5 and 10.0. The conditions used to generate the 4monopegylated IFN samples for analysis were 1) SC-PEG₁₂,000 @ pH 6.5; 2)PC-PEG₁₂,000 @ pH 6.5; 3) SC-PEG₁₂,000 (1 pH 10.0; and 4) PC-PEG₁₂,000 @pH 10.0. In each pH 6.5 case, a 3.9 to 1 molar ratio of PEG:IFN wasused. In each pH 10.0 case, a 3.2:1 molar ratio of PEG:IFN was used.These conditions were chosen to evaluate the influence of both reactionpH and linker on the composition of the final product.

The conjugated material from each reaction condition was recovered andtested for biological activity (CPE assay) and for distribution ofpositional isomers using Mini-S chromatography assay.

The PEG-IFN generated by reacting IFN with PC-PEG₁₂,000 at pH 6.5 hadlower biological activity than that made with SC-PEG₁₂,000 in spite ofboth reagents forming urethane bonds. Thus, it was shown that in spiteof the similarity between the linkers, SC-PEG, anoxycarbonyl-oxy-N-dicarboximide-activated polymer, more preferentiallyattaches to His34. Interestingly, however, the PEG-IFN productsgenerated by carrying out the reaction at pH 10 with both PC-PEG₁₂,000and SC-PEG₁₂,000 had similar biological activities. In both cases,however, the activities were lower than that obtained for SC-PEG₁₂,000at pH 6.5.

Mini-S chromatography assays showed that histidine-34-linked PEG-IFN isthe major positional isomer present when using SC-PEG₁₂,000 at pH 6.5.Lysine-121-linked PEG-IFN is the major positional isomer present whenthe reaction is carried out at pH 6.5 using PC-PEG₁₂,000. At pH 10,Lysine-121-linked PEG-IFN is the major product using either reagent. SeeTable 8.

Thus, the use of acidic pH and anoxycarbonyl-oxy-N-dicarboximide-activated polymer, i.e. SC-PEG, produceconjugates which are unique products which cannot be reproduced bysubstituting another urethane bond-forming activated polymer such asPC-PEG₁₂,000 in place of SC-PEG₁₂,000.

The above materials contained less than 5% total di-PEG andmulti-PEG-IFN as indicated by the size-exclusion HPLC assay.

                  TABLE 8                                                         ______________________________________                                        Summary of MiniS Assay Results                                                        PEAK NUMBER - (Area Percent)                                          Sample    1      2      3    4    5    6    7   8                             ______________________________________                                        SC-PEG; pH 6.5                                                                          2.1    63     ND   0.7  11.8 5.6  3.4 13.3                          PC-PEG; pH 6.5                                                                          ND     4.8    9    9.6  33.8 13   3.8 25.9                          SC-PEG; pH 10                                                                           ND     ND     14.8 11.2 57.6 9.5  3.1 3.8                           PC-PEG; pH 10                                                                           ND     ND     9.6  13.8 51.7 13.7 3.5 7.8                           ______________________________________                                         ND: not detected                                                              Peak Assignment:                                                              Peak 2: His34 linked PEGIFN ;                                                 Peak 4: Lys31 linked PEGIFN;                                                  Peak 5: Lys121 linked PEGIFN;                                                 Peak 6: Lys49 linked PEGIFN;                                                  Peak 7: Lys83 Linked PEGIFN;                                                  Peak 8: Nterminus (cysteine) linked PEGIFN                               

EXAMPLE 13 Cation Exchange Chromatography Characterization

In this example, analytical separation of several batches of PEG-IFNproduct produced using the procedure of Example 11 (pH 6.5) was carriedout using cation exchange chromatography in order to determine the sitesof polymer attachment and identify the individual positional isomers.The cation exchange apparatus was a Mini-S column (Pharmacia Biotech).Mobile phase A included 10 mM sodium acetate pH 5.3 buffer and 25%2-propanol. Mobile phase B contained 500 mM sodium chloride dissolved inmobile phase A. The flow rate was set at 0.5 ml/min and the elutedprotein was detected at 214 nm. The individual PEG-IFN solutions werediluted with 10 mM sodium acetate pH 5.3, containing 2-propanol (5%) to1 mg/ml protein concentration. Injection volumes ranged from 10 to 30μl, depending upon the protein concentration. The following lineargradient was used:

    ______________________________________                                        Time                                                                          (min)           A(%)   B(%)                                                   ______________________________________                                        0               100    0                                                      5               93     7                                                      50              83     17                                                     60              0      100                                                    65              0      100                                                    66              100    0                                                      75              100    0                                                      ______________________________________                                    

The results are provided in Table 9 below and graphically illustrated inFIG. 2.

                  TABLE 9                                                         ______________________________________                                        Area Percent Quantification of PEG-IFN Batches by Cation Exchange             Chromotography                                                                             Peaks                                                            Batch                                                                              Peak 2  3/4     Peak 5                                                                              Peak 6                                                                              Peak 7a                                                                             Peak 7b                                                                             Peak 8                           ______________________________________                                        1    2.6     53.2    5.3   14.2  6.5   3.4   17.2                             2    1.5     54.7    3.3   12.6  6.1   3.2   18.6                             3    1.6     55.3    2.4   11.9  5.5   3.2   20.1                             4    1.7     55.1    2.6   11.6  5.3   3.1   20.5                             5    1.7     54.3    2.7   11.8  5.6   3.2   20.7                             6    1.7     54.5    2.6   11.8  5.3   2.9   21.1                             7    1.9     54.2    2.3   11.6  5.2   3.2   21.5                             ______________________________________                                    

Main Peak Assignment: Peak 2: Lys-134 linked EPG-IFN; Peak 3/4: His-34linked PEG-IFN; Peak 6: Lys-121 linked PEG-IFN and Lys-131 linkedPEG-IFN; Peak 8: Cys-1 linked PEG-IFN.

These results illustrate that a majority of the conjugates were found inpeaks 3 and 4 (His-34 linked PEG-IFN). The results also show thatcontrary to what was expected, most of the conjugates were formed byattaching the polymer to a histidine rather than one of the lysine aminogroups.

Other embodiments of the invention will be apparent to one skilled inthe art from a consideration of this specification or practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with the true scope and spiritof the invention being indicated by the following claims.

What is claimed is:
 1. A pharmaceutical composition, comprising amixture of alpha-interferon-conjugate positional isomers, wherein one ofsaid positional isomers comprises an alpha-interferon covalentlyconjugated to a substantially non-antigenic alkyl terminatedpolyalkylene oxide at a histidine residue on said alpha interferon, sothat said alpha interferon conjugates have a T_(max) at least about 4times greater than unmodified alpha-interferon measured under the sameconditions.
 2. The pharmaceutical composition of claim 1, wherein saidalpha interferon conjugates have a T_(max) at least about 8 timesgreater than unmodified alpha-interferon measured under the sameconditions.
 3. A pharmaceutical composition, comprising a mixture ofalpha-interferon-conjugate positional isomers, wherein one of saidpositional isomers comprises an alpha-interferon covalently conjugatedto a substantially non-antigenic alkyl terminated polyalkylene oxide ata histidine residue on said alpha interferon, so that said alphainterferon conjugates have an AUC at least about 10 times that ofunmodified alpha-interferon measured under the same conditions.
 4. Thepharmaceutical composition of claim 1, wherein said polyalkylene oxideis polyethylene glycol.
 5. The pharmaceutical composition of claim 1,wherein said alpha interferon conjugates have at least about 10% of theactivity of unmodified alpha-interferon measured under the sameconditions when said pharmaceutical composition is administeredsubcutaneously in a mammal.
 6. The pharmaceutical composition of claim3, wherein said alpha interferon conjugates have at least about 10% ofthe activity of unmodified alpha-interferon measured under the sameconditions when said pharmaceutical composition is administeredsubcutaneously in a mammal.
 7. The pharmaceutical composition of claim1, wherein said alpha interferon conjugates have at least about 10% ofthe activity of unmodified alpha-interferon measured under the sameconditions, and a T_(1/2), of about 5 hours alpha phase when saidconjugate is administered subcutaneously in a mammal.
 8. Thepharmaceutical composition of claim 3, wherein saidalpha-interferon-conjugates have at least about 10% of the activity ofunmodified alpha-interferon measured under the same conditions, and aT_(1/2), of about 5 hours alpha phase when said pharmaceuticalcomposition is administered subcutaneously in a mammal.
 9. Thepharmaceutical composition of claim 1, wherein said alpha interferon isinterferon alpha 2b.
 10. The pharmaceutical composition of claim 9,wherein said histidine residue is His34.
 11. The pharmaceuticalcomposition of claim 1, wherein said mixture of said alpha interferonconjugate positional isomers comprises at least about 3 positionalisomers.
 12. The pharmaceutical composition of claim 1 wherein saidmixture of said alpha interferon positional isomers comprises at leastabout 6 positional isomers.
 13. The pharmaceutical composition of claim1, wherein said mixture of said alpha interferon conjugate positionalisomers comprises at least about 8 positional isomers.
 14. Thepharmaceutical composition of claim 13, wherein said alpha interferon isalpha interferon 2b and said mixture of alpha interferon conjugatepositional isomers comprises said polyalkylene oxide linked to saidalpha interferon 2b, at an amino acid residue selected from the groupconsisting of Cys1, Lys31 His34, Lys49, Lys83, Lys121, Lys131, andLys134.
 15. The pharmaceutical composition of claim 4, wherein saidpolyalkylene oxide is a monomethoxy-polyethylene glycol, (mPEG).
 16. Thealpha-interferon-conjugate of claim 1 wherein said polyalkylene oxide isterminated with a C₁₋₄ alkyl.
 17. The pharmaceutical composition ofclaim 1, wherein said polyalkylene oxide has a molecular weight of fromabout 200 to about 35,000.
 18. A pharmaceutical composition, comprisinga mixture of alpha interferon polymer conjugate positional isomers,wherein one of said positional isomers comprises an alpha interferoncovalently conjugated to a substantially non-antigenic polymer at ahistidine residue on said alpha interferon, wherein said substantiallynon-antigenic polymer is selected from the group consisting ofpolypropylene glycol, dextran, polyvinyl pyrrolidones, polyacryl amides,polyvinyl alcohols and carbohydrate-based polymers, so that saidinterferon conjugates have a T_(max) at least about 4 times greater thanunmodified alpha-interferon measured under the same conditions.
 19. Analpha interferon-containing composition, comprising a plurality of alphainterferon polymer conjugates, wherein at least about 15% of theconjugates include covalent attachment of a substantially non-antigenicalkyl terminated polyalkylene oxide at a histidine of said alphainterferon, so that said interferon conjugates have a T_(max) at leastabout 4 times greater than unmodified alpha-interferon measured underthe same conditions.
 20. The alpha interferon-containing composition ofclaim 19, wherein the alpha interferon portion of said alphainterferon-containing composition is alpha interferon 2b and saidhistidine is His34.
 21. The alpha interferon-containing composition ofclaim 19, wherein at least about 30% of said conjugates include covalentattachment of said alkyl terminated polyalkylene oxide at histidine-34of said alpha interferon.
 22. A pharmaceutical composition comprising amixture of alpha interferon 2b-polymer positional isomers, wherein fromabout 30 to about 60% of the positional isomers include a substantiallynon-antigenic alkyl terminated polyalkylene oxide conjugated to theHis34 of said alpha interferon, from about 7 to about 20% of thepositional isomers include a substantially non-antigenic alkylterminated polyalkylene oxide conjugated to the Cys1 of said alphainterferon and about 7 to about 15% of the positional isomers include asubstantially non-antigenic alkyl terminated polyalkylene oxideconjugated to the Lys121 of said alpha interferon, so that saidinterferon conjugates have a T_(max) at least about 4 times greater thanunmodified alpha-interferon measured under the same conditions.